The effort to sequence the human genome has identified around 30,000 genes, each of which represents a blueprint for producing proteins. Scientists would like to understand not only what each protein does, but also how it does it. Where do we start? As visual creatures, we stand to learn much from a protein?s three-dimensional structure. The field of structural biology, fueled by advances in X-ray and NMR methods, continues to determine three-dimensional protein structures at an impressive rate. In the past year, the number of structures deposited in the Protein Data Bank (PDB) has grown by approximately 20%, and as of September 22, 2005, the PDB contained more than 27,000 proteins structures, of which 6,000 are characterized as enzymes. These static structures typically depict a protein in its resting state; however, enzymes are dynamic molecules whose conformation evolves as it executes its designed function. Consequently, to fully understand how an enzyme functions at the molecular level, it is crucial to know the structural changes that ensue as it executes its function. With this knowledge, researchers will be better poised to rationally engineer proteins and peptides with therapeutic value. We recently developed and continue to refine the method of picosecond time-resolved X-ray crystallography. This technique exploits the pump-probe method where a laser pulse (pump) triggers a reaction in a protein crystal and a delayed X-ray pulse (probe) takes a ?snapshot? of the protein?s structure. The sequence of time-resolved snapshots can be stitched together into a movie that allows us to literally ?watch? a protein as it functions. The ID09B time-resolved beamline at the European Synchrotron and Radiation Facility (ESRF) in Grenoble, France is still the only site in the world capable of acquiring time-resolved macromolecular structures with 150-ps time resolution and < 2-? spatial resolution. The ESRF operates in a mode that is optimized for these studies approximately 2 weeks out of each year, and this limited amount of beam time has hindered progress in this field. We are partnering with the Advanced Photon Source (APS) in Argonne, IL to develop picosecond time-resolved capabilities on the BioCARS beamline. When completed (estimated completion date: January 2007), the amount of beamtime available for picosecond time-resolved crystallography will expand significantly. In our studies of ligand migration in myoglobin (Mb), we use a laser pulse to trigger dissociation of CO from the heme binding site, and then use an X-ray pulse to probe the time-resolved structure of the protein. We identified numerous sites in which CO becomes transiently trapped, and observed correlated motion of the protein side chains. When a single point mutation was introduced in a position near the binding site (L29F), the departure of CO from the primary docking site was significantly accelerated. Dramatic differences in the correlated protein displacements in wild-type vs. L29F Mb were found, which provided a structural explanation for these kinetic differences. The structures acquired reflect the average structure of an ensemble of intermediates, not a single molecule. To understand mechanistically how the protein functions, we would prefer to watch the structural evolution of a single protein molecule with picosecond time resolution and atomic spatial resolution. Unfortunately, this dream is experimentally impossible. On the other hand, a computer can model the structural evolution of a single protein via molecular dynamics (MD) simulations. With MD simulations, you get out only what you put in. How do we know whether the equations used to describe the molecular motions are accurate enough to provide a credible description of a protein as it functions? Our time-resolved X-ray structures are acquired on a time and length scale that is accessible to MD simulations, and these data can serve to validate those simulations. In collaboration with Dr. Gerhard Hummer (LCP), we compared ensemble-averaged MD simulations of the L29F mutant of myoglobin with time-resolved X-ray structures. We found that the simulations reproduced the direction, amplitude, and timescales of crystallographically-determined structural changes. This close agreement between theory and experiments at comparable resolution in space and time validated the individual MD trajectories. From numerous single-molecule trajectories, we identified and structurally characterized a conformational switch that directs dissociated ligands to one of two nearby protein cavities. The simulations also helped to assign features in the time-resolved X-ray structures that were weak and partially obscured by the motion of neighboring side chains [Hummer, Schotte and Anfinrud, PNAS (2004)]. In addition to our studies of Mb, we succeeded in acquiring time-resolved X-ray structures of photoactive yellow protein (PYP) on the picosecond time scale [Ihee et al., PNAS (2005)]. We continue to develop and refine a software package, called TReX, that is capable of analyzing time-resolved Laue diffraction images in real time. Dr. Eric Henry (LCP) has been assisting us with this project by developing code to automate and/or speed up several critical steps in our data processing algorithm. ?Real-time? feedback will help us make much more efficient use of the limited and precious beam time available for this research. Time-resolved spectroscopy continues to provide a critical component of our research program. Time-resolved spectroscopic methods have been used to probe protein-ligand interactions at a high level of detail [Lim and Anfinrud, Methods Mol. Biol. (2005)]. We are also using time-resolved spectroscopy to characterize the photophysics of chromophores. With such information, we are able to design optimal protocols for photoactivating proteins studied by time-resolved X-ray methods. In addition to crystallographic methods, we are also pursuing X-ray methods capable of probing structural dynamics in solutions [Anfinrud and Schotte, Science (2005)]. This combination of time-resolved spectroscopic, crystallographic, and computational tools pave the way to explore functionally-important structure transitions at an atomistic level, from which a far more meaningful mechanistic description of protein function will be achieved.